Auditory Training Reverses Lead (Pb)-Toxicity-Induced Changes in Sound-Azimuth Selectivity of Cortical Neurons.

Lead (Pb) causes significant adverse effects on the developing brain, resulting in cognitive and learning disabilities in children. The process by which lead produces these negative changes is largely unknown. The fact that children with these syndromes also show deficits in central auditory processing, however, indicates a speculative but disturbing relationship between lead-exposure, impaired auditory processing, and behavioral dysfunction. Here we studied in rats the changes in cortical spatial tuning impacted by early lead-exposure and their potential restoration to normal by auditory training. We found animals that were exposed to lead early in life displayed significant behavioral impairments compared with naïve controls while conducting the sound-azimuth discrimination task. Lead-exposure also degraded the sound-azimuth selectivity of neurons in the primary auditory cortex. Subsequent sound-azimuth discrimination training, however, restored to nearly normal the lead-degraded cortical azimuth selectivity. This reversal of cortical spatial fidelity was paralleled by changes in cortical expression of certain excitatory and inhibitory neurotransmitter receptor subunits. These results in a rodent model demonstrate the persisting neurotoxic effects of early lead-exposure on behavioral and cortical neuronal processing of spatial information of sound. They also indicate that attention-demanding auditory training may remediate lead-induced cortical neurological deficits even after these deficits have occurred.

An Integrative Approach to Neuroinflammation in Psychiatric disorders and Neuropathic Pain.

Neuroinflammation is a complex process involving both the peripheral circulation and the Central Nervous System (CNS) and is considered to underlie many CNS disorders including depression, anxiety, schizophrenia, and pain. Stressors including early-life adversity, psychosocial stress, and infection appear to prime microglia toward a pro-inflammatory phenotype. Subsequent inflammatory challenges then drive an exaggerated neuroinflammatory response involving the upregulation of pro-inflammatory mediators that is associated with CNS dysfunction. Several pharmacologic inhibitors of pro-inflammatory cytokines including TNF-α and IL-1ß show good clinical efficacy in terms of ameliorating neuroinflammatory processes. Mind/body and plant-based interventions such as yoga, breathing exercises, meditation, and herbs/spices have also been demonstrated to reduce pro-inflammatory cytokines and have a positive impact on depression, anxiety, cognition, and pain. As the intricate connections between the immune system and the nervous system continue to be elucidated, successful therapies for reducing neuroinflammation will likely involve an integrated approach combining drug therapy with nonpharmacologic interventions.

The Ayurvedic plant Bacopa monnieri inhibits inflammatory pathways in the brain.

ETHNOPHARMACOLOGICAL RELEVANCE: Bacopa monnieri (L) Wettst (common name, bacopa) is a medicinal plant used in Ayurveda, the traditional system of medicine of India, as a nootropic. It is considered to be a "medhya rasayana", an herb that sharpens the mind and the intellect. Bacopa is an important ingredient in many Ayurvedic herbal formulations designed to treat conditions such as memory loss, anxiety, poor cognition and loss of concentration. It has also been used in Ayurveda to treat inflammatory conditions such as arthritis. In modern biomedical studies, bacopa has been shown in animal models to inhibit the release of the pro-inflammatory cytokines TNF-α and IL-6. However, less is known regarding the anti-inflammatory activity of Bacopa in the brain. AIM OF THE STUDY: The current study examines the ability of Bacopa to inhibit the release of pro-inflammatory cytokines from microglial cells, the immune cells of the brain that participate in inflammation in the CNS. The effect of Bacopa on signaling enzymes associated with CNS inflammatory pathways was also studied. MATERIALS AND METHODS: Various extracts of Bacopa were prepared and examined in the N9 microglial cell line in order to determine if they inhibited the release of the proinflammatory cytokines TNF-α and IL-6. Extracts were also tested in cell free assays as inhibitors of caspase-1 and matrix metalloproteinase-3 (enzymes associated with inflammation) and caspase-3, which has been shown to cleave protein Tau, an early event in the development of Alzheimer's disease. RESULTS: The tea, infusion, and alkaloid extracts of bacopa, as well as Bacoside A significantly inhibited the release of TNF-α and IL-6 from activated N9 microglial cells in vitro. In addition, the tea, infusion, and alkaloid extracts of Bacopa effectively inhibited caspase 1 and 3, and matrix metalloproteinase-3 in the cell free assay. CONCLUSIONS: Bacopa inhibits the release of inflammatory cytokines from microglial cells and inhibits enzymes associated with inflammation in the brain. Thus, Bacopa can limit inflammation in the CNS, and offers a promising source of novel therapeutics for the treatment of many CNS disorders.

Pb exposure prolongs the time period for postnatal transient uptake of 5-HT by murine LSO neurons.

Pb exposure is associated with cognitive deficits including Attention Deficit Hyperactivity Disorder (ADHD) in children and alters auditory temporal processing in humans and animals. Serotonin has been implicated in auditory temporal processing and previous studies from our laboratory have demonstrated that developmental Pb decreases expression of serotonin (5-HT) in the adult murine lateral superior olive (LSO). During development, certain non-serotonergic sensory neurons, including auditory LSO neurons, transiently take up 5-HT through the serotonin reuptake transporter (SERT). The uptake of 5-HT is important for development of sensory systems. This study examines the effect of Pb on the serotonergic system in the LSO of the early postnatal mouse. Mice were exposed to moderate Pb (0.01mM) or high Pb (0.1mM) throughout gestation and postnatal day 4 (P4) and P8. We found that Pb exposure prolongs the normal developmental expression of 5-HT by LSO neurons and this is correlated with expression of SERT on LSO cell bodies. The prolonged expression of 5-HT by postnatal LSO neurons is correlated with decreased synaptic immunolabeling within the LSO. This Pb-associated decrease in synaptic density within the LSO could contribute to the auditory temporal processing deficits and cognitive deficits associated with developmental Pb exposure.

Chronic low-level Pb exposure during development decreases the expression of the voltage-dependent anion channel in auditory neurons of the brainstem.

Lead (Pb) exposure is a risk factor for neurological dysfunction. How Pb produces these behavioral deficits is unknown, but Pb exposure during development is associated with auditory temporal processing deficits in both humans and animals. Pb disrupts cellular energy metabolism and efficient energy production is crucial for auditory neurons to maintain high rates of synaptic activity. The voltage-dependent anion channel (VDAC) is involved in the regulation of mitochondrial physiology and is a critical component in controlling mitochondrial energy production. We have previously demonstrated that VDAC is an in vitro target for Pb, therefore, VDAC may represent a potential target for Pb in the auditory system. In order to determine whether Pb alters VDAC expression in central auditory neurons, CBA/CaJ mice (n=3-5/group) were exposed to 0.01mM, or 0.1mM Pb acetate during development via drinking water. At P21, immunohistochemistry reveals a significant decrease for VDAC in neurons of the Medial Nucleus of the Trapezoid Body. Western blot analysis confirms that Pb results in a significant decrease for VDAC. Decreases in VDAC expression could lead to an upregulation of other cellular energy producing systems as a compensatory mechanism, and a Pb-induced increase in brain type creatine kinase is observed in auditory regions of the brainstem. In addition, comparative proteomic analysis shows that several proteins of the glycolytic pathway, the phosphocreatine circuit, and oxidative phosphorylation are also upregulated in response to developmental Pb exposure. Thus, Pb-induced decreases in VDAC could have a significant effect on the function of auditory neurons.

Decreased expression of the voltage-dependent anion channel in differentiated PC-12 and SH-SY5Y cells following low-level Pb exposure.

Lead (Pb) has been shown to disrupt cellular energy metabolism, which may underlie the learning deficits and cognitive dysfunctions associated with environmental Pb exposure. The voltage-dependent anion channel (VDAC) plays a central role in regulating energy metabolism in neurons by maintaining cellular ATP levels and regulating calcium buffering, and studies have shown that VDAC expression is associated with learning in mice. In this study, we examined the effect of 5 and 10microM Pb on VDAC expression in vitro in order to determine whether Pb alters VDAC expression levels in neuronal cell lines. PC-12 and SH-SY5Y cells were used since they differentiate to resemble primary neuronal cells. VDAC expression levels were significantly decreased 48 h after exposure to Pb in both cell lines. In contrast, exposure to 24 h of hypoxia failed to produce a decrease in VDAC, suggesting that decreased VDAC expression is not a general cellular stress response but is a result of Pb exposure. This decreased VDAC expression was also correlated with a corresponding decrease in cellular ATP levels. Real-time reverse transcription-polymerase chain reaction demonstrated a significant decrease in messenger RNA levels for the VDAC1 isoform, indicating that Pb reduces transcription of VDAC1. These results demonstrate that exposure to 5 and 10microM Pb reduces VDAC transcription and expression and is associated with reduced cellular ATP levels.

Chronic low-level lead exposure affects the monoaminergic system in the mouse superior olivary complex.

Low-level lead (Pb) exposure is associated with behavioral and cognitive dysfunction, but it is not clear how Pb produces these behavioral changes. Pb has been shown to alter auditory temporal processing in both humans and animals. Auditory temporal processing occurs in the superior olivary complex (SOC) in the brainstem, where it is an important component in sound detection in noisy environments and in selective auditory attention. The SOC receives a serotonergic innervation from the dorsal raphe, and serotonin has been implicated in auditory temporal processing within the brainstem and inferior colliculus. Because Pb exposure modulates auditory temporal processing, the serotonergic system is a potential target for Pb. The current study was undertaken to determine whether developmental Pb exposure preferentially changes the serotonergic system within the SOC. Pb-treated mice were exposed to no Pb, very low Pb (0.01 mM), or low Pb (0.1 mM) throughout gestation and through 21 days postnatally. Brainstem sections from control and Pb-exposed mice were immunostained for the vesicular monoamine transporter 2 (VMAT2), serotonin (5-HT), and dopamine-beta-hydroxylase (DbetaH; a marker for norepinephrine) in order to elucidate the effect of Pb on monoaminergic input into the SOC. Sections were also immunolabeled with antibodies to vesicular glutamate transporter 1 (VGLUT1), vesicular gamma-aminobutyric acid (GABA) transporter (VGAT), and vesicular acetylcholine transporter (VAChT) to determine whether Pb exposure alters the glutaminergic, GABAergic, or cholinergic systems. Pb exposure caused a significant decrease in VMAT2, 5-HT, and DbetaH expression, whereas VGLUT1, VGAT, and VAChT showed no change. These results provide evidence that Pb exposure during development alters normal monoaminergic expression in the auditory brainstem.

Afferent deprivation elicits a transcriptional response associated with neuronal survival after a critical period in the mouse cochlear nucleus.

The mechanisms underlying enhanced plasticity of synaptic connections and susceptibilities to manipulations of afferent activity in developing sensory systems are not well understood. One example is the rapid and dramatic neuron death that occurs after removal of afferent input to the cochlear nucleus (CN) of young mammals and birds. The molecular basis of this critical period of neuronal vulnerability and the transition to survival independent of afferent input remains to be defined. Here we used microarray analyses, real-time reverse transcription PCR, and immunohistochemistry of the mouse CN to show that deafferentation results in strikingly different sets of regulated genes in vulnerable [postnatal day (P)7] and invulnerable (P21) CN. An unexpectedly large set of immune-related genes was induced by afferent deprivation after the critical period, which corresponded with glial proliferation over the same time frame. Apoptotic gene expression was not highly regulated in the vulnerable CN after afferent deprivation but, surprisingly, did increase after deafferentation at P21, when all neurons ultimately survive. Pharmacological activity blockade in the eighth nerve mimicked afferent deprivation for only a subset of the afferent deprivation regulated genes, indicating the presence of an additional factor not dependent on action potential-mediated signaling that is also responsible for transcriptional changes. Overall, our results suggest that the cell death machinery during this critical period is mainly constitutive, whereas after the critical period neuronal survival could be actively promoted by both constitutive and induced gene expression.

Lead exposure during development results in increased neurofilament phosphorylation, neuritic beading, and temporal processing deficits within the murine auditory brainstem.

Low-level lead (Pb) exposure is a risk factor for learning disabilities, attention deficit hyperactivity disorder (ADHD), and other neurological dysfunction. It is not known how Pb produces these behavioral deficits, but low-level exposure during development is associated with auditory temporal processing deficits in an avian model, while hearing thresholds remain normal. Similar auditory processing deficits are found in children with learning disabilities and ADHD. To identify cellular changes underlying this functional deficit, Pb-induced alterations of neurons and glia within the mammalian auditory brainstem nuclei were quantified in control and Pb-exposed mice at postnatal day 21 by using immunohistochemistry, Western blotting, and 2D gel electrophoresis. Pb-treated mice were exposed to either 0.1 mM (low) or 2 mM (high) Pb acetate throughout gestation and through 21 days postnatally. Pb exposure results in little change in glial proteins such as glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), or F4/80 as determined by Western blot analysis and immunohistochemistry. In contrast, Pb exposure alters neuronal structural proteins by inducing increased phosphorylation of both the medium (NFM) and high-weight (NFH) forms of neurofilament within auditory brainstem nuclei. Axons immunolabeled for neurofilament protein show neuritic beading following Pb exposure both in vivo and in vitro, suggesting that Pb exposure also impairs axonal transport. Functional assessment shows no significant loss of peripheral function, but does reveal impairments in brainstem conduction time and temporal processing within the brainstem. These results provide evidence that Pb exposure during development alters axonal structure and function within brainstem auditory nuclei.

Transitional angiogenesis and vascular remodeling during coronary angiogenesis in response to myocardial infarction.

Coronary artery disease (CAD) is a major source of morbidity and mortality in the industrialized world. CAD causes ischemia as a prelude to angina, myocardial infarction and heart failure as specific forms of heart disease causing a decline in the quality of life. CAD or atherosclerosis and the resulting myocardial ischemia trigger a natural angiogenic response that generates collateral circulations. The long-term goal for these studies is to develop therapeutic angiogenesis that augments the natural coronary angiogenesis. This project makes use of an infarcted transgenic mouse model to characterize formation of those collateral circulations in the post-infraction heart. The experiments utilized thoracotomy and a microcauterizer to produce an infarct in transgenic mice and this stimulated neovascularization and allowed labeling of the coronary vessels, thereby defining the morphogenic processes involved in formation of collateral circulations. The results show that the heart consistently responds to infarcts with angiogenesis at 1d post-treatment (PT) that undergoes transition into vascular remodeling at 7d PT with complete remodeling at 14d PT. The vascular remodeling appears to mitigate any net increase in perfusion that may be achieved early in coronary angiogenesis. The results suggest that therapeutic approaches need to shift from an exclusive focus on stimulating angiogenesis to include modulation of vascular remodeling for increased long-term myocardial perfusion.

Motheaten (me/me) mice deficient in SHP-1 are less susceptible to focal cerebral ischemia.

We have demonstrated previously that the protein tyrosine phosphatase SHP-1 seems to play a role in glial development and is upregulated in non-dividing astrocytes after injury. The present study examines the effect of loss of SHP-1 on the CNS response to permanent focal ischemia. SHP-1 deficient (me/me) mice and wild-type littermates received a permanent middle cerebral artery occlusion (MCAO). At 1, 3, and 7 days after MCAO, infarct volume, neuronal survival and cell death, gliosis, and inflammatory cytokine levels were quantified. SHP-1 deficient me/me mice display smaller infarct volumes at 7 days post-MCAO, increased neuronal survival within the ischemic penumbra, and decreased numbers of cleaved caspase 3+ cells within the ischemic core compared with wild-type mice. In addition, me/me mice exhibit increases in GFAP+ reactive astrocytes, F4-80+ microglia, and a concomitant increase in the level of interleukin 12 (IL-12) over baseline compared with wild-type. Taken together, these results demonstrate that loss of SHP-1 results in greater healing of the infarct due to less apoptosis and more neuronal survival in the ischemic core and suggests that pharmacologic inactivation of SHP-1 may have potential therapeutic value in limiting CNS degeneration after ischemic stroke.

The effect of lead on the avian auditory brainstem.

Lead (Pb) continues to be a significant environmental toxin and remains an integral part of many industrial processes, hobbies, and tobacco smoke. Pb has been shown to be a potent toxin to the CNS and low levels of Pb (below the CDC established toxic blood level of 10 microg/dl) have been correlated with decreases in the IQ of children. Pb exposure is a risk factor for dyslexia, and significantly, dyslexics have deficits in auditory temporal processing, including backward masking and amplitude modulation detection. Importantly, Pb-exposed children have been found to be deficient in various aspects of auditory temporal processing, including backward masking. Auditory temporal information is vital for appropriate speech detection and it is not known where within the auditory axis temporal processing takes place, nor is it understood how Pb exposure modifies the cells of the auditory system. To address these questions, we have developed an animal model of auditory temporal processing using chickens and have established that Pb exposure during development results in deficits in backward masking in avians. The current study was undertaken to identify the cellular changes induced by Pb exposure in the auditory brainstem of chickens that are likely anatomical correlates of the observed deficits in backward masking. We found Pb exposure had no effect on neuron number or glial cells within the auditory brainstem. However, Pb exposure does result in significant decreases in the amount of the medium weight neurofilament protein (NFM) as well as decreased NFM phosphorylation within the axons connecting auditory nuclei in the avian brainstem. Because the amount of neurofilament can affect the conduction velocities of axons, these results may provide an anatomical link between Pb exposure, auditory temporal processing deficits, and dyslexia.

Lipopolysaccharide-activated SHP-1-deficient motheaten microglia release increased nitric oxide, TNF-alpha, and IL-1beta.

Accumulating evidence suggests a deleterious role for activated microglia in facilitating neuronal death by producing neurocytotoxic substances during injury, infection, or neurodegenerative diseases. After cochlear ablation, abnormal microglial activation accompanied by increased neuronal loss within the auditory brainstem occurs in motheaten (me/me) mice deficient in the protein tyrosine phosphatase SHP-1. To determine whether abnormally activated microglia contribute to neuronal death in me/me mice, primary microglial cultures from me/me and wild-type mouse cortices were stimulated by the bacterial endotoxin lipopolysaccharide (LPS) to evaluate the secretion of the neurotoxic mediators nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta). Me/me microglia release significantly greater amounts of all three mediators compared with wild-type microglia. However, the increased release of these compounds in microglia lacking SHP-1 does not appear to occur through activation of extracellular signal-regulated kinase (ERK), p38 kinase subgroups of mitogen-activated protein (MAP) kinases, or increases in NF-kappaB-inducing kinase (NIK). These results suggest that abnormal microglial activation and release of neurotoxic compounds may potentiate neuronal death in deafferented cells and can thus potentiate neurodegeneration in the me/me brainstem. Our data also indicate that SHP-1 is engaged in signaling pathways in LPS-activated microglia, but not through regulation of the ERK and p38 MAP kinases.

Adeno-associated virus-mediated gene transfer to hair cells and support cells of the murine cochlea.

More than 28 million Americans suffer from various forms of hearing loss. The lack of effective treatments for many forms of hearing disorders has prompted interest in the potential application of gene delivery techniques to treat both inherited and pathological hearing disorders. However, to develop a gene therapy strategy that will successfully treat hearing disorders, appropriate vectors that are capable of transducing cochlear hair cells and support cells must be identified. In the present study, we examined the efficiency with which AAV vectors (serotypes 1, 2, and 5) transduce hair cells and support cells in cochlear explants from P0 and E13 mice. We further examined the ability of the CBA and GFAP promoters to drive expression of a GFP marker gene in hair cells and support cells. Robust GFP expression was observed in hair cells and support cells following transduction of primary murine cochlear explants with AAV serotypes 1 and 2, but not serotype 5. The CBA promoter predominantly drove GFP expression in hair cells. In contrast, strong expression from the GFAP promoter was observed primarily in support cells. Thus, using AAV vectors and specific promoters, cell-type-specific expression of transgenes can be established within the cochlea.

Loss of SHP-1 phosphatase alters cytokine expression in the mouse hindbrain following cochlear ablation.

Inflammatory cytokines in the central nervous system are largely modulated by glial cells and influence neuronal responses to CNS injury. The protein tyrosine phosphatase SHP-1, an intracellular regulator of many cytokine signaling pathways, has been implicated in mediating the activation of glia. There is a direct correlation between abnormally activated microglia and neuron loss within the SHP-1 deficient motheaten (me/me) mouse auditory brainstem after afferent injury. In order to determine whether loss of SHP-1 creates an aberrant cytokine environment driving the abnormal activation of me/me microglia, the expression of interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) was examined by enzyme-linked immunosorbent assay (ELISA). Normal uninjured me/me mice showed lower IL-10 but higher IL-1beta levels compared to wild-type. Following unilateral cochlear ablation, there is decreased expression of IL-4 and IL-10 in me/me brains compared to wild-type, but IL-1beta is significantly increased. These findings indicate that decreases in anti-inflammatory cytokines, in combination with increased expression of the pro-inflammatory cytokine IL-1beta, may initiate a robust inflammatory reaction within the me/me brain contributing to the neuronal degeneration in the deafferented me/me auditory brainstem. SHP-1 may therefore play a role in limiting CNS inflammation following injury and disease.

Cochlear ablation in mice lacking SHP-1 results in an extended period of cell death of anteroventral cochlear nucleus neurons.

Cochlear ablation results in the death of anteroventral cochlear nucleus (AVCN) neurons from birth to approximately postnatal day 14 (P14) in the murine brainstem. It is not known whether microglial activation contributes to AVCN neuronal death following deafferentation. In order to determine whether microglial activation helps to define the period of neuronal susceptibility within AVCN, we performed unilateral cochlear ablation on mice lacking the protein tyrosine phosphatase SHP-1 (me/me). These mice have been shown to have an exaggerated microglial response following ischemic injury. In the present study, the glial and neuronal response to deafferentation within AVCN was examined in wild-type and me/me mice at P5, P14, and P21. Lack of SHP-1 results in robust microglial but not astrocyte activation within the ablated P14 me/me AVCN. These mice also exhibit approximately 28% neuronal death at P14, a time when normal wild-type littermate controls show little cell death. Glial activation and neuronal loss at P5 and P21 were similar between the two phenotypes, suggesting a role of activated microglia in inducing neuronal death beyond P14 but not P21. These results indicate that activated microglia may participate in determining whether neurons in AVCN live or die following deafferentation.

Focal cerebral ischemia upregulates SHP-1 in reactive astrocytes in juvenile mice.

The role of the tyrosine phosphatase SHP-1 in the hematopoietic system has been well studied; however, its role in the central nervous system (CNS) response to injury is not well understood. Previous studies in our laboratory have demonstrated increased immunoreactivity for SHP-1 in a subset of reactive astrocytes that do not appear to enter the cell cycle following deafferentation of the chicken auditory brainstem. In order to determine whether mammalian astrocytes also upregulate SHP-1 immunoreactivity following CNS injury, a mouse model of focal cerebral ischemia was utilized to study SHP-1 expression. The brains of 3-week-old mice were analyzed at four time points following permanent middle cerebral artery occlusion (MCAO): 1, 3, 7, and 14 days. Our results demonstrate consistent infarct volumes within surgical groups, and infarct volumes decrease as a function of time from 1 day (maximum infarct volume) to 14 days (minimum infarct volume) post-MCAO. In addition, SHP-1 protein levels are upregulated following cerebral ischemia and this increase peaks at 7 days post-MCAO. Analysis of confocal images further reveals that immunoreactivity for SHP-1 occurs predominantly in GFAP+ reactive astrocytes, although a small percentage of F4-80+ microglia are also double labeled for SHP-1 at early times post-MCAO. These SHP-1+ reactive astrocytes do not appear to enter the cell cycle (as defined by PCNA immunoreactivity), confirming our previous studies in the avian auditory brainstem. These results suggest that SHP-1 plays an important role in the regulation of glial activation and proliferation in the ischemic CNS.

SHP-1 expression in avian mixed neural/glial cultures.

The central nervous system response to injury includes astrocyte proliferation and hypertrophy as well as microglial activation and proliferation. However, not all glial cells enter the cell cycle following damage, and the mechanism that determines which glial cells will proliferate and which will remain quiescent has yet to be elucidated. Protein tyrosine phosphorylation has been shown to play an important role in the regulation of the cell cycle in a number of different systems and has been implicated in both astrocyte proliferation and differentiation. Of particular interest is the protein tyrosine phosphatase SHP-1 (Src homology 2-containing protein tyrosine phosphatase 1), which: (1) modulates cellular proliferation in the hematopoietic system, (2) is involved in various growth factor second messenger signaling cascades, and (3) has been demonstrated by our laboratory to increase in immunoreactivity within a subpopulation of astrocytes following deafferentation of the chicken auditory brainstem. These SHP-1+ cells appear to be those which fail to enter the cell cycle following deafferentation. The present study examines whether manipulation of cellular proliferation in vitro modifies the expression of SHP-1 immunoreactivity in mixed neural/glial cultures of the avian auditory brainstem. In addition, the effect of the protein tyrosine phosphatase inhibitor sodium orthovanadate on cellular proliferation was assessed in these cultures. Our results demonstrate that SHP-1 expression can be modulated by changes in proliferation and that inhibiting tyrosine phosphatase activity results in increased proliferation. Taken together, these results indicate that SHP-1 may play central role in negatively regulating glial proliferation following injury.